Epimorphin antagonist and process for producing it

ABSTRACT

This invention provides an epimorphin antagonist comprising part of the functional domain of epimorphin (e.g., a polypeptide specified by the amino acid sequence: 
     H-Ser(Asn)-Gly-Asn-Arg-Thr-Ser-Val-Asp-Leu-Arg-Ile-Arg-Arg-Thr-Gln-His-Ser-Val-Leu-OH SEQ ID NO: 2 and SEQ ID NO: 3) and having the activity of substantially inhibiting the morphogenic activity of epimorphins on epithelial cells, and provides a pharmaceutical containing the epimorphin antagonist as the active ingredient.

This is a division of application Ser. No. 08/981,012, filed Feb. 11,1998, now U.S. Pat. No. 5,998,579 which is a 371 of PCT/JP97/01414 filedApr. 24, 1997.

TECHNICAL FIELD

This invention relates to polypeptides which have the activity ofspecifically inhibiting epimorphins (hereinafter referred to asepimorphin antagonist activity), substances promoting the morphogenesisof epithelial tissue, and which are useful as the active ingredient ofpharmaceuticals.

BACKGROUND ART

The normal morphogenesis of epithelial tissue has been suggested to becontrolled by factors derived from mesenchymal cells present around theepithelial tissue. Diseases ascribed to the abnormal morphogenesis ofepithelial tissue are largely due to abnormalities in mesenchymal cells.These findings have aroused an interest in the clarification of themechanism by which mesenchymal cells control the morphogenesis ofepithelial tissue. However, substances involved in the control ofmorphogenesis of epithelial tissue are expressed under time and spatialcontrol in a complicated system. It is extremely difficult to isolatethese substances and analyze their functions. It is also difficult toconstruct a model experimental system simplifying the morphogenesis ofepithelial tissue. For these and other reasons, much progress has notbeen made to date in researches in this field. Thus, analysis of thecontrol mechanism for the morphogenesis of epithelial tissue has beendemanded keenly in order to elucidate the mechanism of occurrence ofdiseases associated with the morphogenesis of epithelial tissue andestablish methods for treating these diseases.

Under these circumstances, a report was made of success in the isolationof epimorphin taking part in the control of the morphogenesis ofepithelial tissue (Japanese Laid-Open Patent Publication No. 25295/94 orEuropean Patent Application Laid-Open Publication No. 562123,hereinafter called EPA562123). This substance was shown to be aphysiologically active substance containing a protein of 277 to 289amino acids as the core protein, and to be biosynthesized mainly bymesenchymal cells. Epimorphin was also shown to have the action ofpromoting the morphogenesis of epithelial tissue, and it was alsoindicated that when epimorphin did not function, normal tissue formationdid not take place.

As a substance known to inhibit the physiological action of epimorphin(epithelial tissue morphogenesis promoting action), there is an antibody(M-1) which binds specifically to epimorphin, inhibiting its action(Japanese Laid-Open Patent Publication No. 25295/94 or EPA562123; CELL,69, pp. 471-481, 1992). This antibody is useful for clarifying themechanism for the normal morphogenesis of epithelial tissue byepimorphin, and is expected to be useful in the elucidation of themechanism of occurrence of diseases ascribed to the abnormal expression(excessive expression) of epimorphin and in the prevention and treatmentof these diseases. Epimorphin also acts to promote the morphogenesis ofa hair follicle. Thus, the above-mentioned antibody that inhibits theactivity of epimorphin is likely to induce alopecia or inhibit hairgrowth.

However, this antibody posed the problem of being unstable and becomingeasily deactivated during preparation, thus making it difficult todevelop or stably supply it as a pharmaceutical or a hair growthinhibitor. Since its molecular weight is large (more than 150,000 Kd),moreover, it was unable to pass through the keratin of the skin easilyand was not expected to inhibit hair growth sufficiently. For thesereasons, it has been demanded to develop a substance which inhibits theaforementioned activity of epimorphin, and which is stable and has a lowmolecular weight of, say, less than several thousands permitting itspassage through the skin. In particular, epimorphin antagonist, asubstance binding to the receptor on an epithelial cell where epimorphinbinds, and being capable of inhibiting the epithelial tissue morphogenicactivity of epimorphin, is expected to take marked effect in serving asthe active ingredient of a pharmaceutical or a hair growth inhibitor,and in elucidating the mechanism of action of epimorphin.

In addition, in removing hair on the arm or leg, it has been customarypractice to directly pull it out by means of a tape or forceps, or toapply to the site a solution containing an ingredient such as an alkalifor lysis of the hair. The former method causes pain, while the lattermethod damages the skin. If it is possible to provide a substance actingto inhibit the function of epimorphin and having a molecular weight lowenough for percutaneous absorption, therefore, such a substance isexpected to be useful as the active ingredient of a hair growthinhibitor that avoids the above problems. That is, hair growth can besuppressed for a long term by applying a substance with suchcharacteristics after shaving. This substance is usable as a hair growthinhibitor which overcomes the drawbacks of a conventional hair remover.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a substance forinhibiting the action of epimorphin. More particularly, the inventionaims to provide a substance useful as an epimorphin antagonist whichspecifically inhibits the morphogenic activity of epimorphin onepithelial tissue. Further, the invention aims at providing anepimorphin antagonist having such a characteristic effect, a lowmolecular weight, and stability.

Another object of the invention is to provide a pharmaceutical whichcontains as the active ingredient an epimorphin antagonist inhibitingthe morphogenic activity of epimorphin on epithelial tissue, and whichis useful for the prophylaxis and/or treatment of diseases attributableto the excessive expression of epimorphin.

Still another object of the invention is to provide a hair growthinhibitor which contains as the active ingredient the above-describedepimorphin antagonist.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photomicrograph of the histological shape of a mousepulmonary primordium at the start of culture when its organ culture wasperformed in the presence of the epimorphin antagonist of the presentinvention;

FIG. 2 is a photomicrograph of the histological shape after 3 days ofculture when the organ culture of the mouse pulmonary primordium wasperformed in the presence of the epimorphin antagonist of the presentinvention;

FIG. 3 is a photomicrograph of the histological shape of a mousepulmonary primordium at the start of culture when its organ culture wasperformed in the absence of the epimorphin antagonist of the presentinvention;

FIG. 4 is a photomicrograph of the histological shape after 3 days ofculture when the organ culture of the mouse pulmonary primordium wasperformed in the absence of the epimorphin antagonist of the presentinvention;

FIG. 5 is a photomicrograph of the histological shape of a mousemaxillary skin at the start of culture when its organ culture wasperformed in the presence of the epimorphin antagonist of the presentinvention;

FIG. 6 is a photomicrograph of the histological shape after 3 days ofculture when the organ culture of the mouse maxillary skin was performedin the presence of the epimorphin antagonist of the present invention;

FIG. 7 is a photomicrograph of the histological shape of a mousemaxillary skin at the start of culture when its organ culture wasperformed in the absence of the epimorphin antagonist of the presentinvention; and

FIG. 8 is a photomicrograph of the histological shape after 3 days ofculture when the organ culture of the mouse maxillary skin was performedin the absence of the epimorphin antagonist of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

To attain the aforementioned objects, the inventors have conductedextensive studies, finding that a partial sequence polypeptide containedin the functional domain of epimorphin has the activity of markedlyinhibiting the morphogenic activity of epimorphin on epithelial cellsand functions as an epimorphin antagonist. The present invention hasbeen accomplished based on these findings.

In more detail, the invention provides an epimorphin antagonist havingepimorphin antagonist activity and being a polypeptide comprising thefollowing amino acid sequence:

H-X1-Gly-Asn-Arg-Thr-Ser-Val-Asp-Leu-Arg-Ile-Arg-Arg-Thr-Gln-His-Ser-Val-Leu-OHwhere X1 represents Ser or Asn. (SEQ ID NO: 2 and SEQ ID NO: 3)

    1               5                  10                 15H-Ser-Gly-Asn-Arg-Thr-Ser-Val-Asp-Leu-Arg-Ile-Arg-Arg-Thr-Gln-             19  20                  25                  30His-Ser-Val-Leu-Ser-Arg-Lys-Phe-Val-Glu-Ala-Met-Ala-Glu-Tyr-                 35                  40                  45Asn-Glu-Ala-Gln-Thr-Leu-Phe-Arg-Glu-Arg-Ser-Lys-Gly-Arg-Ile-                 50                  55                  60Gln-Arg-Gln-Leu-Glu-Ile-Thr-Gly-Arg-Thr-Thr-Thr-Asp-Asp-Glu-                 65                  70                  75Leu-Glu-Glu-Met-Leu-Glu-Ser-Gly-Lys-Pro-Ser-Ile-Phe-Thr-Ser-                 80          83 Asp-Ile-Ile-Ser-Asp-Ser-Gln-Ile-OH

The invention also provides an epimorphin antagonist having epimorphinantagonist activity and being a polypeptide having the following aminoacid sequence ranging from the 1st amino acid to the nth amino acid (ndenotes an integer of 20 to 83):

H-Asn-Cly-Asn-Arg-Thr-Ser-Val-Asp-Leu-Arg-Ile-Arg-Arg-Thr-Gln-His-Ser-Val-Leu-Ser-Arg-Lys-Phe-Val-Asp-Val-Met-Thr-Glu-Tyr-Asn-Glu-Ala-Gln-Ile-Leu-Phe-Arg,-Glu-Arg-Ser-Lys-Gly-Arg-Ile-Gln-Arg-Gln-Leu-Glu-Ile-Thr-Gly-Arg-Thr-Thr-Thr-Asp-Asp-Glu-Leu-Glu-Glu-Met-Leu-Glu-Ser-Gly-Lys-Pro-Ser-Ile-Phe-Ile-Ser-Asp-Ile-Ile-Ser-Asp-Ser-Gln-Ile-OH(SEQ ID NO:5).

 1                5                  10                  15Asn-Gly-Asn-Arg-Thr-Ser-Val-Asp-Leu-Arg-Ile-Arg-Arg-Thr-Gln-             19  20                  25                  30His-Ser-Val-Leu-Ser-Arg-Lys-Phe-Val-Asp-Val-Met-Thr-Glu-Tyr-                 35                  40                  45Asn-Glu-Ala-Gln-Ile-Leu-Phe-Arg-Glu-Arg-Ser-Lys-Gly-Arg-Ile-                 50                  55                  60Gln-Arg-Gln-Leu-Glu-Ile-Thr-Gly-Arg-Thr-Thr-Thr-Asp-Asp-Glu-                 65                  70                  75Leu-Glu-Glu-Met-Leu-Glu-Ser-Gly-Lys-Pro-Ser-Ile-Phe-Ile-Ser-                 80          83 Asp-Ile-Ile-Ser-Asp-Ser-Gln-Ile

The invention also provides an epimorphin antagonist comprising theabove-described amino acid sequence from which, in which or to which oneto several amino acids have been deleted, replaced or added, and alsohaving epimorphin antagonist activity.

The invention also provides an epimorphin antagonist which comprises apolypeptide containing the above-described amino acid sequence as apartial sequence thereof, and which substantially has epimorphinantagonist activity.

The invention also provides an epimorphin antagonist comprising theabove-described amino acid sequence in which one to several amino acidshave been modified with sugars or lipids, and also having epimorphinantagonist activity.

The invention also provides an epimorphin antagonist comprising theabove-described amino acid sequence in which one to several amino acidshave been phosphorylated, and also having epimorphin antagonistactivity.

The invention also provides an epimorphin antagonist whose epimorphinantagonist activity inhibits the morphogenic activity of epimorphins onepithelial tissue.

The invention also provides an epimorphin antagonist whose epimorphinantagonist activity inhibits the morphogenic activity of epimorphins ona hair follicle.

The invention also provides a pharmaceutical composition containing theabove-described epimorphin antagonist as the active ingredient.

The invention also provides the above-described pharmaceuticalcomposition for use in the treatment and/or prevention of diseaseascribed to the excessive expression of epimorphin.

The invention also provides the above-described pharmaceuticalcomposition in which the disease ascribed to the excessive expression ofepimorphin is hyperepimorphinism.

The invention also provides the above-described pharmaceuticalcomposition which is used as a hair growth inhibitor.

The invention also provides a polynucleotide encoding the aforementionedpolypeptide.

The above-described polypeptides provided by the invention have theactivity of inhibiting the morphogenic activity of epimorphins onepithelial cells. Thus, the present invention provides epimorphinantagonists comprising the polypeptides. The term “epimorphins” in thepresent specification is used to express a concept including naturallyoccurring epimorphin, a modified form of the natural type epimorphinthat has substantially the same physiological activity as the naturalepimorphin (i.e. modified epimorphin), and amino acid variants of thenatural type epimorphin and the modified epimorphin that havesubstantially the same physiological activity as the natural epimorphin.The epimorphin antagonist of the present invention has the activity ofinhibiting the morphogenic activity of any substances selected from thegroup consisting of the natural type epimorphin, the modifiedepimorphin, and their amino acid variants.

In the present specification, the natural type epimorphin refers toepimorphin biosynthesized by mesenchymal cells of, say, mammals.Examples of the natural type epimorphin include epimorphin derived fromanimals such as human, monkey, cattle, horse, sheep, dog, cat, rabbit,rat and mouse, preferably, human-derived epimorphin. Among the naturaltype epimorphin may be a plurality of isoforms produced by geneticsplicing. Among the human epimorphins, for instance, there are humanepimorphin consisting of 288 amino acids, and isoforms A and B of humanepimorphin composed of 287 and 277 amino acids, respectively, asdescribed in Japanese Laid-Open Patent Publication No. 25295/94. Themouse epimorphin includes mouse epimorphin consisting of 289 aminoacids, and isoforms A and B of mouse epimorphin composed of 288 and 279amino acids, respectively. The term “natural type epimorphin” in thepresent specification is used to express a concept including all ofthese isoforms.

In the present specification, the modified epimorphin refers to apolypeptide which has substantially the same physiological activity asthe natural type epimorphin (e.g., cell adhesion to epithelial cells andmorphogenesis promotion for epithelial cells) and which either is apartial polypeptide sequence derived from the polypeptide sequence ofthe natural type epimorphin (normally a polypeptide comprising 277 to289 amino acids), or contains as its partial sequence the partialpolypeptide sequence derived from the polypeptide sequence of thenatural type epimorphin. The natural type epimorphin has a coiled coilarea (1) on the N-terminal side, a functional domain (2) in the middle,a coiled coil area (3) on the C-terminal side, and a hydrophobic area atthe C-terminal, and binds to the cell membrane at the C-terminalhydrophobic area. A method for removing the C-terminal hydrophobic areahas been proposed with a view to solubilizing the natural typeepimorphin (Japanese Laid-Open Patent Publication No. 25295/94). Apolypeptide produced by such a method is a typical compound falling inthe category of the modified epimorphin.

In the present specification, the amino acid variant of the natural typeepimorphin or the modified epimorphin refers to a polypeptide which hassubstantially the same physiological activity as the natural typeepimorphin and in which one or more of the amino acids constituting thepolypeptide chain of the natural type epimorphin or the modifiedepimorphin have been replaced by other amino acids, or in which one ormore of these constituent amino acids have been deleted, and/or in whichone or more arbitrary amino acids have been inserted into thispolypeptide chain.

More concretely, the natural type epimorphin is disclosed, for example,in Japanese Laid-Open Patent Publication No. 25295/94 as human- ormouse-derived epimorphin and isoforms thereof. Typical examples of themodified epimorphin are, in addition to the above-mentioned modifiedepimorphin (Japanese Laid-Open Patent Publication No. 25295/94 orEPA562123), modified epimorphin produced by adding a hydrophilicpolypeptide comprising 5 to 99 amino acids to at least one end of apolypeptide containing the functional domain of epimorphin (JapaneseLaid-Open Patent Publication No. 325293/96 or European PatentApplication Laid-Open Publication No. 698666 (hereinafter calledEPA698666)), and modified epimorphin comprising a polypeptide of thestructure in which the hydrophobic area at the C-terminal has beendeleted from the entire length of the epimorphin having the coiled coilarea (1) on the N-terminal side, functional domain (2) in the middle,coiled coil area (3) on the C-terminal side, and hydrophobic area at theC-terminal, and in which at least some amino acids have been deletedfrom at least one terminal side of the coiled coil areas (1) and (3)(Japanese Laid-Open Patent Publication No. 65885/97 or EPA698666).

It should be understood that besides all the modified epimorphinsdisclosed in the above-quoted publications and specifications, modifiedepimorphins producible by the methods disclosed in these publicationsand specifications, or changed or modified forms of these methods, areall included in the modified epimorphin in the present specification aslong as they fulfill the aforementioned definitions. Methods forproducing the amino acid variant of the natural type epimorphin or themodified epimorphin are explained concretely, for example, in JapaneseLaid-Open Patent Publication No. 325293/96 and Japanese Laid-Open PatentPublication No. 65885/97 or EPA698666. However, they are not restrictedto these methods, and amino acid variants produced by any methods areavailable. The testing of the physiological activity of these modifiedepimorphins or their amino acid variants can be performed in accordancewith the testing method for the physiological activity of the naturaltype epimorphin that has been described in detail in Japanese Laid-OpenPatent Publication No. 25295/94 or EPA562123. Concretely, it suffices toconfirm the morphogenic activity like branching morphogenesis of a mousefetal lung or a mouse fetal skin morphogenesis that is described in theExamples of the present specification.

The epimorphin antagonist of the present invention has the feature ofinhibiting the morphogenic activity of the above-described epimorphinson epithelial cells (hereinafter, this feature may be referred to hereinas “epimorphin antagonist activity”). As the epimorphin antagonist ofthe present invention, there is provided an epimorphin antagonist whichcomprises part of the functional domain of epimorphin and substantiallyhas the epimorphin antagonist activity. As an embodiment thereof,polypeptide (I) is provided which is specified by the following aminoacid sequence:

H-Ser(Asn)-Gly-Asn-Arg-Thr-Ser-Val-Asp-Leu-Arg-Ile-Arg-Arg-Thr-Gln-His-Ser-Val-Leu-OH

(where the amino acid residue at the N-terminal maybe replaced by theparenthesized amino acid residue, each amino acid residue preferablyrepresents an L-amino acid residue; this sequence is designated asH-S(N)GNRTSVDLRIRRTQHSVL-OH according to one-letter designation) (SEQ IDNO: 2 and SEQ ID NO: 3).

The polypeptide (I) is a partial sequence of the functional domain ofepimorphin (an amino acid sequence ranging from the 104th to 187th aminoacids counting from the N-terminal in the case of natural type humanepimorphin, an amino acid sequence ranging from the 105th to 188th aminoacids counting from the N-terminal in the case of natural type mouseepimorphin; see Japanese Laid-Open Patent Publication No. 325293/96 orEPA698666), and corresponds to an amino acid sequence ranging from the1st to 19th amino acids of the functional domain. This functional domainhas so far been shown to exhibit adhesion to and morphogenesis promotionfor epithelial cells, but has not been reported to show epimorphinantagonist activity. Nor is there any report on the production of thispolypeptide.

The epimorphin antagonist of the present invention includes not only thepolypeptide (I), but also an amino acid variant of the polypeptide (I)in which one or more of its constituent amino acids have been replacedby other amino acids, or in which one or more of these constituent aminoacids have been deleted, and/or in which one or more arbitrary aminoacids have been inserted into the polypeptide chain thereof, and whichis a polypeptide substantially having epimorphin antagonist activity.The kinds of the one or more amino acids to replace and/or to beinserted are not restricted, but they are preferably L-amino acids.

The epimorphin antagonist of the present invention includes apolypeptide which contains the polypeptide (I) or its variant as apartial sequence thereof and which substantially has epimorphinantagonist activity. For example, one or more amino acids may be bondedto the N-terminal and/or C-terminal of the polypeptide (I), andpreferably, an arbitrary oligopeptide composed of two or more arbitraryamino acids may be bonded thereto. The kinds of such amino acids are notrestricted, but are preferably selected from L-amino acids. Forinstance, a polypeptide comprising the polypeptide (I) to whoseN-terminal about 1 to 10, preferably 5 to 7, more preferably 6,L-histidines have been linked as a tag sequence is preferred from thepoint of view of purification efficiency.

Epimorphin antagonist as another embodiment of the present invention ispolypeptide (II) specified by an amino acid sequence ranging from the Ist (N-terminal) to the nth (n denotes an integer of 20 to 83) amino acidin the following amino acid sequence (A):

H-Ser(Asn)-Gly-Asn-Arg-Thr-Ser-Val-Asp-Leu-Arg-Ile-Arg-Arg-Thr-Gln-His-Ser-Val-Leu-Ser-Arg-Lys-Phe-Val-Glu(Asp)-Ala(Val)-Met-Ala(Thr)-Glu-Tyr-Asn-Glu-Ala-Gln-Thr(Ile)-Leu-Phe-Arg-Glu-Arg-Ser-Lys-Gly-Arg-Ile-Gln-Arg-Gln-Leu-Glu-Ile-Thr-Gly-Arg-Thr-Thr-Thr-Asp-Asp-Glu-Leu-Glu-Glu-Met-Leu-Glu-Ser-Gly-Lys-Pro-Ser-Ile-Phe-Thr(Ile)-Ser-Asp-Ile-Ile-Ser-Asp-Ser-Gln-Ile-OH

(where the amino acid residue located on the left of the parentheses ( )may be replaced by the parenthesized amino acid residue, and each aminoacid residue preferably represents an L-amino acid residue) (SEQ ID NO:4 and SEQ ID NO: 5). This amino acid sequence corresponds to a sequencespecified by the 1st to 83rd amino acids in the polypeptide sequence ofthe functional domain of epimorphins.

The epimorphin antagonist of the present invention includes not only thepolypeptide (II) selected from the amino acid sequence (A), but also avariant of the polypeptide (II) in which one or more of its constituentamino acids have been replaced by other amino acids, or in which one ormore of these constituent amino acids have been deleted, and/or in whichone or more arbitrary amino acids have been inserted into thepolypeptide chain thereof and, which is a polypeptide substantiallyhaving epimorphin antagonist activity. The kinds of the one or moreamino acids to replace and/or to be inserted are not restricted, butthey are preferably L-amino acids.

The epimorphin antagonist of the present invention also includes apolypeptide which contains the polypeptide (II) or its variant as apartial sequence thereof-and which substantially has epimorphinantagonist activity. For example, one or more amino acids: may be bondedto the N-terminal and/or C-terminal of the polypeptide, and preferably,an oligopeptide of an arbitrary length composed of two or more arbitraryamino acids may be bonded thereto. The kinds of such amino acids are notrestricted, but are preferably selected from L-amino acids. Forinstance, a polypeptide comprising the above polypeptide to whoseN-terminal about 1 to 10, preferably 5 to 7, more preferably 6,L-histidines have been linked as a tag sequence is preferred from thepoint of view of purification efficiency.

However, a polypeptide comprising the polypeptide (II) to whoseC-terminal threonine (Thr) has been linked has been demonstrated to actas the functional domain of the natural type epimorphin. Thus, it is notpreferred that one L-threonine (L-Thr) is linked to the C-terminal ofthe polypeptide (II). For the same reason, it is not preferred that apolypeptide specified by the sequence

H-Ser-Arg-Lys-Phe-Val-Glu(Asp)-Ala(Val)-Met-Ala(Thr)-Glu-Tyr-Asn-Glu-Ala-Gln-Thr(Ile)-Leu-Phe-Arg-Glu-Arg-Ser-Lys-Gly-Arg,-Ile-Gln-Arg-Gln-Leu-Glu-Ile-Thr-Gly-Arg-Thr-Thr-Thr-Asp-Asp-Glu-Leu-Glu-Glu-Met-Leu-Glu-Ser-Gly-Lys-Pro-Ser-Ile-Phe-Thr(Ile)-Ser-Asp-Leu-Ile-Ser-Asp-Ser-Gln-Ile-Thr-OH

(where the amino acid residue located on the left of the parentheses ( )may be replaced by the parenthesized amino acid residue, and each aminoacid residue preferably represents an L-amino acid residue is linked tothe C-terminal of the polypeptide (I)) (SEQ ID NO: 9 and SEQ ID NO: 10).

The polypeptide of the present invention that has been described in eachof the embodiments may be in free form, but may be supplied as an acidadded salt such as hydrochloride, acetate or p-toluenesulfonate, or abase added salt such as ammonium salt or organic amine salt. Thus, thepolypeptide in the present specification should be interpreted asincluding the polypeptide in the form of the salt. Furthermore, theabove polypeptide having arbitrary sugars (monosaccharides,disaccharides, oligosaccharides or polysaccharides) or lipids linkedthereto, and the polypeptide which has been phosphorylated, are alsoincluded in the scope of the epimorphin antagonist of the presentinvention.

The Examples in the present specification concretely describe thetesting methods for typical examples of epimorphin antagonist activity,i.e., the activity of inhibiting the morphogenesis of the bronchus of amouse fetal lung and the activity of inhibiting the morphogenesis of amouse fetal maxillary skin, in regard to the polypeptide (I) as apreferred embodiment of the epimorphin antagonist of the presentinvention. Thus, those skilled in the art can easily make sure, byreference to these testing examples or through a suitable change ormodification of these methods, that each of the polypeptides definedabove has the desired epimorphin antagonist activity. The morphogenesispromoting action of epimorphin on epithelial tissue is described indetail, for instance, in the Examples of Japanese Laid-Open PatentPublication No. 25295/94 or EPA562123. The application of the testingsystem described there enables epimorphin antagonist activity to beconfirmed.

The Examples in the present specification demonstrate the polypeptide(I), a particularly preferred embodiment of the present invention, tohave cell adhesion properties similar to those of epimorphins. Byreferring to those testing examples or adding a suitable change ormodification to those methods, it becomes possible to confirm easilythat each of the polypeptides has cell adhesion properties. Theinventors do not stick to any particular theory, but the epimorphinantagonist of the invention has the property of binding to an epimorphinreceptor present on the extracellular surface of an epithelial cell.This antagonist shows the same cell adhesion as do epimorphins, but uponbinding to the receptor, it has no morphogenesis promoting action asexhibited by epimorphins, or cannot fully show this action. Hence, theepimorphin antagonist of the invention may act as a competitiveinhibitor for epimorphins.

The epimorphin antagonist activity of the epimorphin antagonist of theinvention is not restricted in terms of the potency of its inhibitoryactivity as long as this antagonist can substantially inhibit themorphogenetic action of epimorphins on epithelial tissue. Preferably,however, the antagonist should have epimorphin antagonist activitycomparable, for instance, to that of the polypeptide (I). The Examplesin the present specification describe typical examples of epimorphinantagonist activity. However, the epimorphin antagonist activity is notrestricted to these examples, but should be interpreted more broadly.Any action which inhibits the morphogenesis promoting action ofepimorphins on epithelial cells by any mechanism is, needless to say,included in the epimorphin antagonist activity of the epimorphinantagonist of the present invention.

The epimorphin antagonist of the invention can be synthesized by achemical technique in customary practice for peptide synthesis, such asthe solid phase or liquid phase method. The protective group for anamino group or the like, and the condensation agent for a condensationreaction, in peptide synthesis include, for example, those described inSuzuki, K.: “Protein Engineering—Basis and Application” (1992, MaruzenCo., Ltd.); Pondanski et al.: “Peptide Synthesis” (1976, John Wiley &Sons, N.Y.); and Stewart et al.: “Solid Phase Peptide Synthesis” (1969,W. H. Freeman and Co., San Francisco). With the solid phase method,commercially available various peptide synthesizers can be utilized. TheExamples in the present specification describe a concrete method forproducing the polypeptide (I) by means of a peptide synthesizer.

An ordinary biological method such as a gene expression procedure isalso available. In this case, a recombinant vector containing a DNAsequence encoding the polypeptide chain of the epimorphin antagonist ofthe present invention is used to prepare a microorganism transformed bythe vector (transformant). The desired polypeptide, epimorphinantagonist, can be separated and purified from a culture of thetransformant. Of course, the production method for the epimorphinantagonist of the invention is not restricted to the above-describedmethods.

DNA usable for the method of production by gene expression includes, forexample, DNA specified by the 1st nucleotide to the 57th nucleotide inthe following base sequence:

AGTGGGAACC GGACTTCAGT GGATCTTCGG ATACGAAGAA 40

CCCAGCATTC GGTGCTGTCT CGGAAGTTTG TGGAAGCCAT 80

GGCGGAGTAC AATGAGGCAC AGACTCTGTT TCGGGAGCGG 120

AGCAAAGGCC GCATCCAGCG CCAGCTGGAG ATAACTGGGA 160

GAACCACCAC AGACGACGAG CTAGAAGAGA TGCTGGAGAG 200

CGGGAAGCCA TCCATCTTCA CTTCCGACAT TATATCAGAT 240

TCACAAATT(249) (SEQ ID NO: 1)

or DNA specified by the 1st nucleotide to the (3×kth) nucleotide (kdenotes an integer of 20 to 83) in the above-based sequence (only thesense strand is shown with complimentary base sequence being omitted).This DNA corresponds to the 1 st to 249th nucleotides of DNA comprisingthe base sequence designated as SEQ ID NO: 6 in the Sequence Listingthat encodes a polypeptide as the functional domain of natural typehuman epimorphin.

Similarly, of DNA corresponding to the 1st to 249th nucleotides of DANcomprising the base sequence designated as SEQ ID NO: 7 in the SequenceListing that encodes a polypeptide as the functional domain of naturaltype mouse epimorphin, there can be utilized DNA specified by the 1stnucleotide to the 57th nucleotide; or DNA specified by the 1stnucleotide to the (3×mth) nucleotide (m denotes an integer of 20 to 83).

Using these DNA's, amino acid variants can be easily produced by thecustomary method. Usable as this method is, for example, the recombinantPCR method described on pages 155-160 of “PCR Experimental Manual (1991,HJB Publishing Bureau), or the PCR-based method for preparation of amutant gene described on pages 63-67 of “Experimental Medicine, SpecialIssue, Vol. 8, No. 9” (1990, Shodo-sha). To produce the desiredpolypeptide, the method for gene expression may be, but not restrictedto, the method described in detail in the Examples of Japanese Laid-OpenPatent Publication No. 65885/97 or EPA698666.

The epimorphin antagonist of the present invention is useful against theexcessive expression of various morphogenetic factors typified bynatural type epimorphin, and preferably useful as the active ingredientof a pharmaceutical for the treatment and/or prevention of diseasesassociated with the excessive expression of natural type epimorphin, oras the active ingredient of a pharmaceutical for the diagnosis of suchdiseases. The epimorphin antagonist of the present invention is alsouseful as the active ingredient of a pharmaceutical for the treatment ofhyperepimorphinism induced by epimorphins which are administered for thetreatment and/or prevention of diseases associated with the minimalexpression of natural type epimorphin. Furthermore, the epimorphinantagonist of the present invention is useful as the active ingredientof a pharmaceutical for use as a hair growth inhibitor. The term“pharmaceutical” in the present specification is used in its broadestsense, including ones for use in the prevention, treatment and diagnosisof diseases in mammals including human, as well as hair growthinhibitors which are normally classified as quasi-drugs.

As morphogenetic factors, various ones including natural type epimorphinare suggested to exist. Diseases caused by the excessive expression ofone or more of these factors include, for example, rheumatoid arthritis,carcinoma such as renal carcinoma or skin carcinoma, arteriosclerosis,collagen disease, hematopoietic disease, renal disease, musculardystrophy, osteoporosis, neurofibromatosis, Sturge-Weber syndrome,tuberous sclerosis, impaired neural tube closure, segmental disorder,vagal disorder, callosal agenesis, porencephaly, and hydrocephalus. Thepharmaceutical of the invention can be expected to prove useful for thetreatment and/or prevention, as well as diagnosis, of these diseases.However, the objects to which the pharmaceutical of the invention isapplied to are not restricted to these diseases. It should be understoodthat diseases which may be associated with the excessive expression ofone or more morphogenetic factors, especially epimorphin, are allincluded in the scope of the targets for application of thepharmaceutical of the invention. The uses of a hair growth inhibitorcontaining epimorphin antagonist as the active ingredient, an embodimentof the pharmaceutical of the invention, should be interpreted in thebroadest sense, including those for alopecia, inhibition of hair growth,and inhibition of trichogenesis.

As the pharmaceutical of the invention, one or more substances selectedfrom among epimorphin antagonists which are the aforementionedpolypeptides may be used as such. Normally, however, it is preferredthat one or more pharmaceutically acceptable pharmaceutical additives beused to produce a pharmaceutical composition containing one or more ofthe above substances as the active ingredient, and this composition beused for the treatment and/or prophylaxis of the aforesaid diseases.From the viewpoints of solubility, pharmacokinetics such as absorptionand excretion and/or the production method, those polypeptides may be inthe form of pharmaceutically acceptable salts. The route ofadministration of the pharmaceutical composition includes, for example,systemic administration such as intravenous, rectal or oraladministration, as well as local administration such as externalapplication, ophthalmic instillation, nasal instillation, oticinstillation, or local injection.

For example, drugs for systemic administration such as intravenousinjections or drip infusions, or drugs for local administration such asointments, creams, plasters or local injections are preferred forms ofthe pharmaceutical composition of the present invention. The use of apharmaceutical composition containing the active ingredient encapsulatedin liposome or the like, or a pharmaceutical composition havingantibodies or the like bound thereto may be able to improve its affinityor selectivity for the target organ. Of course, the route ofadministration may be selected suitably according to the type of diseaseto be targeted, the purpose of treatment or prevention, the kind of thelesion, and the condition of the patient. The dosage form preferred forthe route of administration may also be chosen suitably. The form of thepharmaceutical composition when used as a diagnostic reagent is notrestricted, and includes its in vivo administration to the patient andits in vitro use on samples taken from the patient.

A hair growth inhibitor containing one or more substances selected fromthe epimorphin antagonists of the invention as the aforementionedpolypeptides is preferably supplied as a preparation in the formpreferred for the purpose of use as a hair growth inhibitor, such ascream, spray, coating solution, or plaster. The polypeptide may be inthe form of a pharmaceutically acceptable salt. It is also preferredthat a suitable surface active agent or a liposoluble material isblended with its cream in order to allow the efficient percutaneousabsorption of the epimorphin antagonist, i.e., the active ingredient,through the keratin layer of the skin.

EXAMPLES

The present invention will be described further concretely by thefollowing Examples, but the scope of the invention is not limitedthereto:

Example 1 Production of Epimorphin Antagonist of the Invention

A polypeptide having an amino acid sequence of 19 amino acids rangingfrom the 1st (N-terminal) amino acid to the 19th amino acid in thefunctional domain (the amino acid sequence described as SEQ ID NO: 8 inthe Sequence Listing) in the middle of natural type mouse epimorphin waschemically synthesized using a peptide synthesizer (Advanced Chemtech396 Synthesizer). This material was purified by means of reversed phaseHPLC under the following conditions to obtain polypeptide A with apurity of more than 90%.

Column: Reversed phase Perseptive Biosystems Poros R2

Adsorption buffer: 0.1% aqueous solution of trifluoroacetic acid

Elution buffer:

Solution A (0.1% aqueous solution of trifluoroacetic acid)

Solution B (acrylonitrile incorporating 0.1% aqueous solution oftrifluoroacetic acid)

Elution with 76.2% solution A +23.8% solution B in 2.015 minutes.

Similarly, polypeptide B having an amino acid sequence of 39 amino acidsranging from the 1st (N-terminal) amino acid to the 39th amino acid, andpolypeptide C having an amino acid sequence of 41 amino acids rangingfrom the 1st (N-terminal) amino acid to the 41st amino acid, in thefunctional domain (the amino acid sequence described as SEQ ID NO: 8 inthe Sequence Listing) in the middle of natural type mouse epimorphinwere obtained by synthesis and purification by means of the samereversed phase HPLC. Each polypeptide had a purity of more than 90%.

Polypeptide B: Elution with 93.7% solution A+6.3% solution B in 9.27minutes

Polypeptide C: Elution with 93.a% solution A+6.2% solution B in 9.18minutes

Example 2 Production of Modified Epimorphin

cDNA's encoding a fragment of mouse-derived epimorphin deprived of thehydrophobic area at the C-terminal, and the functional domain in themiddle of the epimorphin (fragment (123) and fragment (2), respectively,described in the Example of Japanese Laid-Open Patent Publication No.65885/97 or EPA698666) were prepared by PCR in accordance with themethod described in the Example of Japanese Laid-Open Patent PublicationNo. 65885/97 or EPA698666. Then, they were integrated into the NdeI andNheI sites of pET3C vector having the area between two Eco RV sitesdeleted therefrom to construct an expression vector. Then, thisexpression vector was introduced into E. coli BL21 strain turned intocompetent cells by the Hanahan method (“Lab Manual Genetic Engineering”,Maruzen Co., Ltd.). The introduction of the vector was performed byadding an expression vector solution (1 mg/ml solution, 1 μl) into asolution (100 μl) of the competent cells on ice, allowing the mixture tostand for 10 minutes on ice, leaving this mixture to stand in anincubator for 2 minutes at 42° C., and then allowing the system to standfor 30 minutes on ice.

Then, the E. coli cells were cultured on an LB plate (1% Bacto tryptone,0.5% Bacto-yeast extract, 1% NaCl, 1.5% Bacto-agar) containing 50 μg/mlampicillin, and colonies which were growing were selected. Thesetransformants were examined by the PCR method for DNA encoding thedesired modified epimorphin. It was confirmed that 9 of 10 strains werethe desired transformants retaining the expression vector. The resultingtransformants were mass cultured by 37° C. shake culture in a liquid LBculture medium (1% Bacto tryptone, 0.5% Bacto-yeast extract, 1% NaCl)containing 50 μg/ml ampicillin. Then, IPTG, a material for inducingexpression, was added into the culture medium to a final concentrationof 1 mM. Further, shake culture was continued for 2 hours at 37° C. toexpress modified epimorphins (fragment (123) and fragment (2) in the E.coli cells.

Analysis of the total protein in the E. coli cells by SDS-polyacrylamideelectrophoresis showed that the desired modified epimorphins wereproduced in nearly equal amounts. The above-mentioned transformants thatexpressed the modified epimorphins were suspended and washed in a lysisbuffer [50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 100 mM NaCl], andcentrifuged to precipitate the cells. The cells were suspended in thelysis buffer, and then a lysozyme (SIGMA, L-6876) was added to aconcentration of 1 mg/ml. Freeze-thawing was repeated 3 times to lysethe E. coli. After ultrasonication, the supernatant was removed bycentrifugation. The precipitate was washed 4 times with 2M urea/lysisbuffer. Then, the precipitate was resuspended in 8M urea/lysis buffer,and centrifuged to obtain a supernatant fraction (purity: more than 90%)containing the desired modified epimorphins.

Example 3 Cell Adhesion

The polypeptides A, B and C of the present invention obtained in Example1, and the modified epimorphins (fragment (123) and fragment (2), eachhaving the same cell adhesion capacity as that of natural typeepimorphin) were coated on suspension culture medium dishes. Afterdrying, the dishes were washed once with 8M urea/lysis buffer. Then, thedishes were washed 5 times with PBS⁻, and the cell strainC3H/10T1/2clone8 (Dainippon Pharmaceutical Co., Ltd., 08-226) wassprinkled on the dishes using D-MEM/F-12 culture medium (GIBCO BRL,12400-024; hereinafter called “DH medium”) containing 20 mg/ml bovineserum albumin (SIGMA, A-7030). One hour later, the dishes were washed 3times with PBS⁻, and then the cells were collected using 0.5N NaOH. TheO.D. at 260 nm was measured to determine the number of the cells boundto the dishes. The cell count was used as an indicator of the celladhesion of each specimen. The O.D.'s found were 1.5237 for polypeptideA, 1.4251 for polypeptide B, 1.4697 for polypeptide C, 1.4959 forfragment (123), 1.4478 for fragment (2), and 0.0194 for control(untreated). Thus, polypeptides A, B and C were all confirmed to showcell adhesion comparable to that of the modified epimorphins.

Example 4 Inhibitory Action on Morphogenesis of Bronchus

A pulmonary primordium was removed from the fetus of the ICR pregnantmouse (Nippon Charles River, 12 days of gestation) under a stereoscopicmicroscope. The pulmonary primordium taken was placed on Nucleporemembrane filter (SN 110419) floated on a culture medium (DH medium, or0.35 mg/ml polypeptide A-containing DH medium). Organ culture wasperformed in 5% CO₂ at 37° C. to investigate the effect of polypeptide Aon the morphogenesis of the bronchus. As shown in FIGS. 3 and 4, whenorgan culture used DH medium, the morphogenesis of the bronchus normallyproceeded, and finely branched bronchus formation was confirmed atcompletion of culture. When the pulmonary primordium was organ culturedin the culture medium containing polypeptide A, on the other hand, themorphogenesis of the bronchus was markedly inhibited (FIGS. 1 and 2).This manner of inhibition of morphogenesis was exactly the same as intreatment with MC-1, i.e., antibodies inhibiting the function ofepimorphin. Hence, it is clear that the polypeptide A of the presentinvention inhibits the morphogenetic action of epimorphin on a pulmonaryprimordium.

Example 5 Inhibitory Action on Morphogenesis of Maxillary Skin

A maxillary skin was removed from the fetus of the ICR pregnant mouse(Nippon Charles River, 13 days of gestation) under a stereoscopicmicroscope. The maxillary skin taken was placed on Nuclepore membranefilter (SN 110419) floated on a culture medium (DH medium, or 0.35 mg/mlpolypeptide A-containing DH medium). Organ culture was performed in 5%CO₂ at 37° C. to investigate the effect of polypeptide A on themorphogenesis of the hair follicle. As shown in FIGS. 7 and 8, whenorgan culture used DH medium, the morphogenesis of normal hair folliclesfrom the maxillary skin proceeded, and the formation of a plurality ofhair follicles was confirmed. When the maxillary skin was organ culturedin the culture medium containing polypeptide A, on the other hand, themorphogenesis of hair follicles was markedly inhibited (FIGS. 5 and 6).This manner of inhibition of morphogenesis was exactly the same as intreatment with MC-1, i.e., antibodies inhibiting the function ofepimorphin. Hence, it is clear that the polypeptide A of the presentinvention inhibits the hair follicle morphogenetic action of epimorphinon a maxillary skin.

Industrial Applicability

The epimorphin antagonist of the present invention has the activity ofspecifically inhibiting the morphogenic activity of epimorphins onepithelial tissue. Thus, it is useful as the active ingredient of apharmaceutical for the treatment and/or prevention of diseases ascribedto the excessive expression of epimorphin, or as the active ingredientof a hair growth inhibitor.

10 1 249 DNA Homo sapiens 1 agtgggaacc ggacttcagt ggatcttcgg atacgaagaacccagcattc ggtgctgtct 60 cggaagtttg tggaagccat ggcggagtac aatgaggcacagactctgtt tcgggagcgg 120 agcaaaggcc gcatccagcg ccagctggag ataactgggagaaccaccac agacgacgag 180 ctagaagaga tgctggagag cgggaagcca tccatcttcacttccgacat tatatcagat 240 tcacaaatt 249 2 19 PRT Homo sapiens 2 Ser GlyAsn Arg Thr Ser Val Asp Leu Arg Ile Arg Arg Thr Gln His 1 5 10 15 SerVal Leu 3 19 PRT Mus musculus 3 Asn Gly Asn Arg Thr Ser Val Asp Leu ArgIle Arg Arg Thr Gln His 1 5 10 15 Ser Val Leu 4 83 PRT Homo sapiens 4Ser Gly Asn Arg Thr Ser Val Asp Leu Arg Ile Arg Arg Thr Gln His 1 5 1015 Ser Val Leu Ser Arg Lys Phe Val Glu Ala Met Ala Glu Tyr Asn Glu 20 2530 Ala Gln Thr Leu Phe Arg Glu Arg Ser Lys Gly Arg Ile Gln Arg Gln 35 4045 Leu Glu Ile Thr Gly Arg Thr Thr Thr Asp Asp Glu Leu Glu Glu Met 50 5560 Leu Glu Ser Gly Lys Pro Ser Ile Phe Thr Ser Asp Ile Ile Ser Asp 65 7075 80 Ser Gln Ile 5 83 PRT Mus musculus 5 Asn Gly Asn Arg Thr Ser ValAsp Leu Arg Ile Arg Arg Thr Gln His 1 5 10 15 Ser Val Leu Ser Arg LysPhe Val Asp Val Met Ala Glu Tyr Asn Glu 20 25 30 Ala Gln Ile Leu Phe ArgGlu Arg Ser Lys Gly Arg Ile Gln Arg Gln 35 40 45 Leu Glu Ile Thr Gly ArgThr Thr Thr Asp Asp Glu Leu Glu Glu Met 50 55 60 Leu Glu Ser Gly Lys ProSer Ile Phe Ile Ser Asp Ile Ile Ser Asp 65 70 75 80 Ser Gln Ile 6 252DNA Homo sapiens 6 agtgggaacc ggacttcagt ggatcttcgg atacgaagaacccagcattc ggtgctgtct 60 cggaagtttg tggaagccat ggcggagtac aatgaggcacagactctgtt tcgggagcgg 120 agcaaaggcc gcatccagcg ccagctggag ataactgggagaaccaccac agacgacgag 180 ctagaagaga tgctggagag cgggaagcca tccatcttcacttccgacat tatatcagat 240 tcacaaatta ct 252 7 252 DNA Mus musculus 7aatgggaacc gaacttcagt ggatctgcgg atacgaagga cccagcactc ggtgctgtca 60cggaagtttg tggacgtcat gacagaatac aatgaagcgc agatcctgtt ccgggagcga 120agcaaaggcc gcatccagcg ccagctggag atcactggga ggaccaccac tgacgacgag 180ctggaagaga tgctggagag cgggaagccg tccatcttca tctcggatat tatatcagat 240tcacaaatca ct 252 8 84 PRT Mus musculus 8 Asn Gly Asn Arg Thr Ser ValAsp Leu Arg Ile Arg Arg Thr Gln His 1 5 10 15 Ser Val Leu Ser Arg LysPhe Val Asp Val Met Ala Glu Tyr Asn Glu 20 25 30 Ala Gln Ile Leu Phe ArgGlu Arg Ser Lys Gly Arg Ile Gln Arg Gln 35 40 45 Leu Glu Ile Thr Gly ArgThr Thr Thr Asp Asp Glu Leu Glu Glu Met 50 55 60 Leu Glu Ser Gly Lys ProSer Ile Phe Ile Ser Asp Ile Ile Ser Asp 65 70 75 80 Ser Gln Ile Thr 9 65PRT Homo sapiens 9 Ser Arg Lys Phe Val Glu Ala Met Ala Glu Tyr Asn GluAla Gln Thr 1 5 10 15 Leu Phe Arg Glu Arg Ser Lys Gly Arg Ile Gln ArgGln Leu Glu Ile 20 25 30 Thr Gly Arg Thr Thr Thr Asp Asp Glu Leu Glu GluMet Leu Glu Ser 35 40 45 Gly Lys Pro Ser Ile Phe Thr Ser Asp Ile Ile SerAsp Ser Gln Ile 50 55 60 Thr 65 10 65 PRT Mus musculus 10 Ser Arg LysPhe Val Asp Val Met Thr Glu Tyr Asn Glu Ala Gln Ile 1 5 10 15 Leu PheArg Glu Arg Ser Lys Gly Arg Ile Gln Arg Gln Leu Glu Ile 20 25 30 Thr GlyArg Thr Thr Thr Asp Asp Glu Leu Glu Glu Met Leu Glu Ser 35 40 45 Gly LysPro Ser Ile Phe Ile Ser Asp Ile Ile Ser Asp Ser Gln Ile 50 55 60 Thr 65

What is claimed is:
 1. An isolated polynucleotide encoding an epimorphinantagonist having epimorphin antagonist activity, wherein the epimorphinantagonist is a polypeptide consisting of amino acid sequence SEQ ID NO:2 or SEQ ID NO:
 3. 2. An isolated polynucleotide encoding an epimorphinantagonist having epimorphin antagonist activity, wherein the epimorphinantagonist is a polypeptide consisting of the amino acid sequence setforth in SEQ ID NO: 4 or the amino acid sequence of the 1st to the nthamino acid of SEQ ID NO: 4, wherein n denotes an integer of 20 to
 83. 3.An isolated polynucleotide encoding an epimorphin antagonist havingepimorphin antagonist activity, wherein the epimorphin antagonist is apolypeptide consisting of the amino acid sequence set forth in SEQ IDNO: 5 or the amino acid sequence of the 1st to the nth amino acid of SEQID NO: 5, wherein n denotes an integer of 20 to 83.